Streptococcus pyogenes secretes several proteins to the extracellular environment that directly influence host-pathogen interactions and contribute to virulence. Many secreted proteins have been studied in detail; however, the functions of several others are unknown. The locus-designated rgg is required for the expression of streptococcal pyrogenic exotoxin B (SPE B), a secreted cysteine protease that contributes to virulence. Inactivation of rgg also altered the expression of additional secreted proteins. Results obtained from genetic and physiological analyses of the rgg mutant strain have led to the hypothesis that the composition of available catabolic substrates influences exoprotein expression in an Rgg-dependent manner. To test the hypothesis, the following aims are proposed: Specific Aim 1. Determine if the availability of nitrogen-containing catabolic substrates influences Rgg dependent expression of virulence-associated exoproteins. Quantitative RT-PCR will be used to identify changes in mf-1 and speB expression in response to the availability of catabolic substrates. Proteomics and metabolite analysis will be used to assess the influence of catabolic substrates on exoprotein expression. Specific Aim 2. Identify Rgg-regulated proteins. Differences in protein expression between wild-type strain NZ131 and an isogenic rgg mutant will be detected with two-dimensional gel electrophoresis and differentially expressed proteins identified with mass spectrometry. Specific Aim 3. Distinguish between Rgg-regulated proteins and changes in expression due to perturbations of other regulatory circuits. Changes in protein expression will be identified with proteomics following induction of rgg expression by using a nisin-inducible promoter. Specific Aim 4. Determine if Rgg binds to promoter regions of genes encoding exoproteins to control expression. Electrophoretic mobility-shift assays will be used to determine if Rgg binds to the promoter regions of mf-1 and speB.